Extracellular phospholipases A2(PLA2) have been suggested to initiate and propagate inflammation in several diseases. A specific inhibitor of this enzyme activity would be an ideal antiinflammatory agent. In an attempt to generate such a specific inhibitor of PLA2 on the basis of its antibody structure, we have produced and fully characterized a monospecific neutralizing antibody against porcine pancreatic PLA2, a prototype for extracellular PLA2s. This antibody virtually completely inhibits PLA2 activity in vitro. It has an IC50 around 6nM. From affinity purified IgG we have isolated F(ab)2 and Fab fragments which inhibit PLA2 activity in a dose dependent manner very similar to that of an intact IgG. An IC50 of 10 to 20 nM was obtained. Epitope mapping by CNBr and V8 protease revealed that a stretch of amino acid residues 21-40 of PLA2 is the most likely epitope recognized by the antibody. This region includes several invariable residues forming part of the calcium binding loop (Tyr 28, Gly 3O and Gly 32) which is essential for PLA2 activity. These data paved the way for generating monoclonal antibodies against the calcium binding loop of PLA2, cloning and expression of Fab in E. coli and for generating specific inhibitors for therapeutic use. In a related study, we demonstrated that TG can catalyze the incorporation of polyamines into PLA2 and dramatically increase the activity of this enzyme. In another investigation we have been successful in expressing high level of human uteroglobin in E. coli and characterized it. This protein is also a PLA2 inhibitor similar to rabbit uteroglobin. Generation of a monospecific antibody of this protein facilitated the characterization. In a collaborative study we have delineated the calcium binding site of human osteopontin, a sialoprotein elaborated by osteoblasts, normal kidney cells, mammary gland and metastatic tumor cells. We also found that the three molecular species (e.g., 62kDa, 69kDa and 75kDa) have identical amino acid sequence and the varying molecular weights are due to the level of glycosylation. This protein appears in human milk in large quantities and may be responsible for calcium transport to the infant. The regulation of this gene may have implications in normal mineralization of bones as well as osteoporosis and other demineralizing diseases. In order to develop a specific inhibitor of HIV-1 aspartic protease (AP) on the basis of its antibody structure, we have generated several monoclonal antibodies directed against oligopeptides corresponding to one amino terminal and one carboxy terminal unique regions of this enzyme. These regions bear no homology to mammalian AP. The characterization of these antibodies is in progress.